PROJECT SUMMARY Successful production of healthy zygotes involves the activity of dozens of genes in both males and females. These genes mediate a complex set of interactions between the sexes at the molecular, cellular, neurological and whole-organism levels. In the current funding period, we identified high levels of naturally occurring variation in genes that act in the female's nervous system to modulate sperm precedence. In this renewal application, we propose as Aim 1 a set of experiments that ask whether conspecific sperm precedence is mediated by the same genes that are selected upon for within-species sperm competition. Mating with males from the wrong species might produce a sperm-selective response through the same mechanisms as the ones that mediate preference among conspecific males, or there may be qualitative differences. This question will be addressed directly by genetic tests of sperm precedence, and by transcriptome comparisons, in within- and between-species matings. We will quantify the transfer of RNAs by male Drosophila, map genes involved in variation in this transfer, and relate RNA transfer to sperm competition outcomes. In Aim 2, we ask whether and how the highly conserved octopamine (OA) neural-signaling pathway is modulated to play a role in male x female sperm-competitive interactions. We show with preliminary results that direct manipulation of OA in females influences differential retention of sperm from the first male to mate, raising the question of how the sexes use this pathway to influence each others' reproductive outcome. We will test this question directly, by genetic manipulation of OA signaling followed by tests of sperm precedence, and by examining variation of OA signaling, and its relationship to sperm competition outcomes, in natural inbred reference lines. In Aim 3 we explore the role of exosomes in mediating male x female interactions. Exosomes are lipid-bound vesicles that carry both proteins and small RNA molecules. They define a new cell-cell communication mechanism with roles in processes as diverse as fertility and metastasis. Exosomes are transferred to females in seminal fluid and impact reproductive phenomena such as sperm maturation in mammals. Similar vesicles in Drosophila seminal fluid fuse with fly sperm, and possibly with the female reproductive tract, affecting female post-mating responses. It is possible to knock out seminal exosome transmission in flies, and to follow the presence, transfer and fusion of these exosomes with a fluorescent marker. We propose using these manipulations to determine whether exosomes transfer signals between males and females that impact sperm use. Our work to examine variation in exosome production and transfer will entail application of GWAS methods to identify new genetic factors in exosome biology. Many of the processes that we will study show high levels of evolutionary conservation, implying that our results will expand our understanding of male x female interactions in reproduction that may have relevance to cases of idiopathic human infertility that may involve genetic incompatibility between the partners.